Tunable indocyanine dyes for biomedical applications

ABSTRACT

The sensitivity and specificity of the optical modality can be enhanced by the use of highly absorbing dyes as contrast agents. Novel indocyanine dyes that absorb and emit light in the near infrared region of electromagnetic spectrum are disclosed. These dyes are useful for imaging, diagnosis and therapy of various diseased states. Particularly, the molecules of the invention are useful for optical diagnostic imaging and therapy, in endoscopic applications for the detection of tumors and other abnormalities, for localized therapy, for photoacoustic tumor imaging, detection and therapy, and for sonofluorescence tumor imaging, detection and therapy.

This is a Divisional Application of U.S. application Ser. No. 09/484,320filed on Jan. 18, 2000.

FIELD OF INVENTION

This invention relates generally to novel cyanine and indocyanine dyesfor use in imaging, diagnosis and therapy. Particularly, this inventionrelates to compositions of cyanine and indocyanine dyes wherein novelcarbocyclic and heterocyclic moieties are incorporated into the polyeneportion of the dye molecules.

BACKGROUND OF THE INVENTION

Several dyes that absorb and emit light in the visible and near-infraredregion of electromagnetic spectrum are currently being used for variousbiomedical applications due to their biocompatibility, high molarabsorptivity, or high fluorescence quantum yields. The high sensitivityof the optical modality in conjunction with dyes as contrast agentsparallels that of nuclear medicine and permits visualization of organsand tissues without the undesirable effect of ionizing radiation.Cyanine dyes with intense absorption and emission in the near-infrared(NIR) region are particularly useful because biological tissues areoptically transparent in this region (B. C. Wilson, Optical propertiesof tissues. Encyclopedia of Human Biology, 1991, 5, 587-597). Forexample, indocyanine green, which absorbs and emits in the NIR regionhas been used for monitoring cardiac output, hepatic functions, andliver blood flow (Y-L. He, H. Tanigami, H. Ueyama, T. Mashimo, and I.Yoshiya, Measurement of blood volume using indocyanine green measuredwith pulse-spectrometry: Its reproducibility and reliability. CriticalCare Medicine, 1998, 26(8), 1446-1451; J. Caesar, S. Shaldon, L.Chiandussi, et al., The use of Indocyanine green in the measurement ofhepatic blood flow and as a test of hepatic function. Clin. Sci. 1961,21, 43-57) and its functionalized derivatives have been used toconjugate biomolecules for diagnostic purposes (R. B. Mujumdar, L. A.Ernst, S. R. Mujumdar, et al., Cyanine dye labeling reagents:Sulfoindocyanine succinimidyl esters. Bioconjugate Chemistry, 1993,4(2), 105-111; Linda G. Lee and Sam L. Woo. “N-Heteroaromatic ion andiminium ion substituted cyanine dyes for use as fluorescent labels”,U.S. Pat. No. 5,453,505; Eric Hohenschuh, et al. “Light imaging contrastagents”, WO 98/48846; Jonathan Turner, et al. “Optical diagnostic agentsfor the diagnosis of neurodegenerative diseases by means of nearinfra-red radiation”, WO 98/22146; Kai Licha, et al. “In-vivo diagnosticprocess by near infrared radiation”, WO 96/17628; Robert A. Snow, etal., Compounds, WO 98/48838).

A major drawback in the use of cyanine dye derivatives is the potentialfor hepatobilliary toxicity resulting from the rapid clearance of thesedyes by the liver (G. R. Cherrick, S. W. Stein, C. M. Leevy, et al.,Indocyanine green: Observations on its physical properties, plasmadecay, and hepatic extraction. J. Clinical Investigation, 1960, 39,592-600). This is associated with the tendency of cyanine dyes to formaggregates in solution which could be taken up by Kupffer cells in theliver. Various attempts to obviate this problem have not been verysuccessful. Typically, hydrophilic peptides, polyethyleneglycol oroligosaccharide conjugates have been used but these resulted inlong-circulating products which are eventually cleared by the liver.Another major difficulty with current cyanine and indocyanine dyesystems is that they offer a limited scope in the ability to inducelarge changes in the absorption and emission properties of these dyes.Attempts have been made to incorporate various heteroatoms and cyclicmoieties into the polyene chain of these dyes (L. Strekowski, M.Lipowska, and G. Patonay, Substitution reactions of a nucleofugal groupin hetamethine cyanine dyes. J. Org. Chem., 1992, 57, 4578-4580; N.Narayanan, and G. Patonay, A new method for the synthesis ofheptamethine cyanine dyes: Synthesis of new near infrared fluorescentlabels. J. Org. Chem., 1995, 60, 2391-2395; E. Fung and R. Rajagopalan,Monocyclic functional dyes for contrast enhancement in optical imaging,U.S. Pat. No. 5,732,104; R. Rajagopalan and E. Fung, Delta^(1,6)bicyclo[4,4,0] functional dyes for contrast enhancement in opticalimaging, U.S. Pat. No. 5,672,333; R. Rajagopalan and E. Fung, Tricyclicfunctional dyes for contrast enhancement in optical imaging, U.S. Pat.No. 5,709,845) but the resulting dye systems do not show largedifferences in absorption and emission maxima, especially beyond 830 nmwhere photacoustic diagnostic applications are very sensitive. They alsopossess prominent hydrophobic core which enhances liver uptake. Further,most cyanine dyes do not have the capacity to form dendrimers which areuseful in biomedical applications.

Therefore, there is a need to design novel dyes that could prevent dyeaggregation in solution, predisposed to form dendrimers, capable ofabsorbing or emitting beyond 800 nm, possess desirable photophysicalproperties, and endowed with tissue-specific targeting capability.

The publications and other materials used herein to support thebackground of the invention or provide additional details respecting thepractice, are incorporated by reference.

SUMMARY OF THE INVENTION

The present invention relates particularly to the novel compositioncomprising cyanine dyes of general formula 1

wherein a₁ and b₁ vary from 0 to 5; W¹ and X¹ may be the same ordifferent and are selected from the group consisting of —CR¹⁰R¹¹, —O—,—NR¹², —S—, and —Se; Q¹ is a single bond or is selected from the groupconsisting of —O—, —S—, —Se—, and —NR¹³; Y¹ and Z¹ may be the same ordifferent and are selected from the group consisting of —(CH₂)_(c)—CO₂H,—CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H, —(CH₂)_(e)—NH₂,—CH₂—(CH₂—O—CH₂)_(f)—CH₂—NH₂, —(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)-CH₂—CO₂H; R¹ and R¹⁰ to R¹⁵ may besame or different and are selected from the group consisting of-hydrogen, C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl, C1-C10polyalkoxyalkyl, —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20 polyhydroxyalkyl,C1-C10 polyhydroxyaryl, —(CH₂)_(d)—CO₂H, —CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H,—(CH₂)_(f)-NH₂, and —CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, and i varyfrom 1 to 10; d, f and j vary from 1 to 100; and R² to R⁹ may be thesame or different and are selected from the group consisting ofhydrogen, C1-C10 alkyl, C1-C10 aryl, hydroxyl, C1-C10 polyhydroxyalkyl,C1-C10 alkoxyl, amino, C1-C10 aminoalkyl, cyano, nitro and halogen.

The present invention also relates to the novel composition comprisingindocyanine dyes of general formula 2

wherein a₂ and b₂ are defined in the same manner as a₁ and b₁; W² and X²are defined in the same manner W¹ and X¹; Q² is defined in the samemanner as Q¹; R¹⁶ and R¹⁰ to R¹⁵ are defined in the same manner as R¹and R¹⁰ to R¹⁵; Y² is defined in the same manner as Y¹; Z² is defined inthe same manner as Z¹; and R¹⁷ to R²⁸ are defined in the same manner asR² to R⁹.

The present invention also relates to the novel composition comprisingcyanine dyes of general formula 3

wherein a₃ and b₃ are defined in the same manner as a₁ and b₁; W³ and X³are defined in the same manner W¹ and X¹; Y³ is defined in the samemanner as Y¹; Z³ is defined in the same manner as Z¹; A₁ is a single ora double bond; if A₁ is a single bond, then B₁ and C₁ may the same ordifferent and are selected from the group consisting of —O—, —S—, —Se—,—P—, and —NR³⁸ and D₁ is selected from the group consisting of —CR³⁹R⁴⁰,and —C═O; if A₁ is a double bond, then B₁ is selected from the groupconsisting of —O—, —S—, —Se—, —P— and —NR³⁸, C₁ is nitrogen or —CR⁴¹,and D₁ is —CR⁴²; R²⁹ to R³⁷ are selected from the group consisting ofhydrogen, C1-C10 alkyl, C1-C10 aryl, hydroxyl, hydrophilic peptide,C1-C10 polyhydroxyalkyl, C1-C10 alkoxyl, cyano, nitro, halogen and—NR⁴³R^(44;) R³⁸ to R⁴² may be same or different and are selected fromthe group consisting of -hydrogen, C1-C10 alkyl, C1-C10 aryl, C1-C10alkoxyl, C1-C10 polyalkoxyalkyl, —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20polyhydroxyalkyl, C1-C10 polyhydroxyaryl, —(CH₂)_(d)—CO₂ H,—CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H, —(CH₂)_(f)—NH₂, and—CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, and i vary from 1 to 10; d, fand j vary from 1 to 100; R⁴³ and R⁴⁴ may be the same or different andare selected from the group consisting of hydrogen, C1-C10 alkyl, C1-C10aryl, or may together form a 5, 6, or 7 membered carbocyclic ring or a5, 6, or 7 membered heterocyclic ring optionally containing one or moreoxygen, nitrogen, or a sulfur atom.

The present invention also relates to the novel composition comprisingindocyanine dyes of general formula 4 wherein a₄ and b₄ are defined inthe same manner as a₁ and b₁; W⁴ and X⁴ are defined in the same manneras W¹ and X¹; Y⁴ is defined in the same manner as Y¹; Z⁴ is defined inthe same manner as Z¹; A₂ is defined in the same manner as A₁; B₂, C₂,and D₂ are defined in the same manner as B₁, C₁, and D₁; and R⁴⁵ to R⁵⁷are defined in the same manner as R²⁹ to R³⁷.

The present invention also relates to the novel composition comprisingcyanine dyes of general formula 5

wherein a₅ is defined in the same manner as a₁; W⁵ and X⁵ are defined inthe same manner W¹ and X¹; Y⁵ is defined in the same manner as Y¹; Z⁵ isdefined in the same manner as Z¹; A₃ is defined in the same manner asA₁; B₃, C₃, and D₃ are defined in the same manner as B₁, C₁, and D₁; andR⁵⁸ to R⁶⁶ are defined in the same manner as R²⁹ to R³⁷.

The present invention also relates to the novel composition comprisingcyanine dyes of general formula 6

wherein a₆ is defined in the same manner as a₁; W⁶ and X⁶ are defined inthe same manner as W¹ and X¹; Y⁶ is defined in the same manner as Y¹; Z⁶is defined in the same manner as Z¹; A₄ is defined in the same manner asA₁; B₄, C₄, and D₄ are defined in the same manner as B₁, C₁, and D₁; andR⁶⁷ to R⁷⁹ are defined in the same manner as R²⁹ to R³⁷.

This invention is also related to the method of conjugating the dyes ofthis invention to peptides or biomolecules by solid phase synthesis.

This invention is also related to the method of preventing fluorescencequenching. It is known that cyanine dyes generally form aggregates inaqueous media leading to fluorescence quenching. We observed that ininstances where the presence of hydrophobic core in the dyes leadfluorescence quenching, the addition of a biocompatible organic solventsuch as 1-50% dimethylsulfoxide (DMSO) restored the fluorescence bypreventing aggregation and allowed in vivo organ visualization.

DETAILED DESCRIPTION OF THE INVENTION

The novel compositions of the present invention comprising dyes offormulas 1 to 6 offer significant advantages over those currentlydescribed in the art. As illustrated in FIGS. 1-6, these dyes aredesigned to prevent aggregation in solution by preventing intramolecularand intermolecular ordered hydrophobic interactions. They also havemultiple attachment sites proximal to the dye chromophore for ease offorming dendrimers. The presence of rigid and extended chromophorebackbone enhances fluorescence quantum yield and extends the maximumabsorption beyond 800 nm. Conjugation of biomolecules to these dyes arereadily achievable. They are useful in various biomedical applicationsincluding, but not limited to, tomographic imaging of organs; monitoringof organ functions; coronary angiography; fluorescence endoscopy;detection, imaging, and therapy of tumors; laser guided surgery,photoacoustic and sonofluorescence methods; and the like. Specificembodiments to accomplish some of the aforementioned biomedicalapplications are given below.

In one embodiment of the invention, the dyes of the invention are usefulfor optical tomographic, endoscopic, photoacoustic and sonofluoresenceapplications for the detection and treatment of tumors and otherabnormalities.

In another aspect of the invention, the dyes of the invention are usefulfor localized therapy.

In yet another aspect of the invention, the dyes of the invention areuseful for the detection of the presence of tumors and otherabnormalities by monitoring the blood clearance profile of the dyes.

In a further embodiment of the invention, the dyes are useful for laserassisted guided surgery for the detection of micrometastases of tumorsupon laparoscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

In yet another aspect of the invention, the dye bioconjugates of thedyes of this invention are useful diagnosis of atherosclerotic plaquesand blood clots.

The novel dyes of the present invention are prepared according themethods well known in the art and are shown in FIGS. 1-5 and their usein the synthesis of bioconjugates is shown in FIG. 6.

FIG. 1 shows the reactions in the synthesis of bis-carboxylates of thisinvention.

FIG. 2 shows the reactions in the synthesis of tetra-carboxylates ofthis invention.

FIG. 3 shows the reactions in the synthesis of polyhydroxy-carboxylicacid cyanine dyes of this invention.

FIG. 4 shows the reactions in the synthesis of non-aggregating cyaninedyes of this invention.

FIG. 5 shows the reactions in the synthesis of “tunable” cyanine dyes ofthis invention.

FIG. 6 shows the reactions in a representative scheme for thepreparation of bioconjugates of this invention.

In a preferred embodiment, the dyes according to the present inventionhave the general Formula 1 wherein a₁ and b₁ vary from 0 to 3; Q1 is asingle bond; R¹ to R⁹ are hydrogens; W¹ and X¹ may be the same ordifferent and are selected from the group consisting of —C(CH₃)₂,C((CH₂)_(zz)OH)CH₃, C((CH₂)_(zz)OH)₂, C((CH₂)_(zz)CO₂H)CH₃,C((CH₂)_(zz)CO₂H)₂, C((CH₂)_(zz)NH₂)CH₃, C((CH₂)_(zz)NH₂)₂,C((CH₂)_(zz)NR_(pp)R_(pz))CH₃ and C((CH₂)_(zz)NR_(pp)R_(pz))₂; Y¹ and Z¹may be the same or different and are selected from the group consistingof —(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R_(pp) and R_(pz) may bethe same or different and are selected from the group consisting of—(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R¹⁴ and R¹⁵ may be sameor different and are selected from the group consisting of -hydrogen,C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl,—CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20 polyhydroxyalkyl, C1-C10polyhydroxyaryl, —(CH₂)_(d)—CO₂H, —CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H,—(CH₂)_(f)NH₂, and —CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, i, and zzvary from 1 to 5; and d, f and j vary from 1 to 100.

In another preferred embodiment, the dyes according to the presentinvention have the general Formula 2 wherein a₂ and b₂ vary from 0 to 3;Q² is a single bond; R¹⁶ to R²⁸ are hydrogens; W² and X² may be the sameor different and are selected from the group consisting of —C(CH₃)₂,C((CH₂)_(zz)OH)CH₃, C((CH₂)_(zz)OH)₂, C((CH₂)_(zz)CO₂H)CH₃,C((CH₂)_(zz)CO₂H)₂, C((CH₂)_(zz)NH₂)CH₃, C((CH₂)_(zz)NH₂)₂,C((CH₂)_(zz)NR_(pp)R_(pz))CH₃and C((CH₂)_(zz)NR_(pp)R_(pz))₂; Y² and Z²may be the same or different and are selected from the group consistingof —(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R_(pp) and R_(pz) may bethe same or different and are selected from the group consisting of—(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R¹⁴ and R¹⁵ may be sameor different and are selected from the group consisting of -hydrogen,C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl,—CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20 polyhydroxyalkyl, C1-C10polyhydroxyaryl, —(CH₂)_(d)—CO₂H, —CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H,—(CH₂)_(f)NH₂, and —CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, i, and zzvary from 1 to 5; and d, f and j vary from 1 to 100.

In another preferred embodiment, the dyes according to the presentinvention have the general Formula 3 wherein a₃ and b₃ vary from 0 to 3;A₁ is a single bond; B₁ is selected from the group consisting of —O—,—S—, and —NR³⁸; C₁ is —CH₂ or —C═O; D₁ is selected from the groupconsisting of —O—, —S—, and —NR³⁸; R²⁹ is a hydrogen, a halogen atom, asaccharide or a hydrophilic peptide; R³⁰ to R³⁷ are hydrogens; R³⁸ isselected from the group consisting of -hydrogen, C1-C10 alkyl, C1-C10aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl,—CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20 polyhydroxyalkyl, C1-C10polyhydroxyaryl, —(CH₂)_(d)—CO₂H, —CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H,—(CH₂)_(f)—NH₂, and —CH₂-(CH₂—O—CH₂)_(g)—CH₂—NH₂; W³ and X³ may be thesame or different and are selected from the group consisting of—C(CH₃)₂, C((CH₂)_(zz)OH)CH₃, C((CH₂)_(zz)OH)₂, C((CH₂)_(zz)CO₂H)CH₃,C((CH₂)_(zz)CO₂H)₂, C((CH₂)_(zz)NH₂)CH₃, C((CH₂)_(zz)NH₂)₂,C((CH₂)_(zz)NR_(pp)R_(pz))CH₃and C((CH₂)_(zz)NR_(pp)R_(pz))₂; Y³ and Z³may be the same or different and are selected from the group consistingof —(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R_(pp) and R_(pz) may bethe same or different and are selected from the group consisting of—(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R¹⁴ and R¹⁵ may be sameor different and are selected from the group consisting of -hydrogen,C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl, C1-C10 polyalkoxyalkyl,—CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20 polyhydroxyalkyl, C1-C10polyhydroxyaryl, —(CH₂)_(d)—CO₂H, —CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H,—(CH₂)_(f)—NH₂, and —CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, i, and zzvary from 1 to 5; and d, f and j vary from 1 to 100.

In another preferred embodiment, the dyes according to the presentinvention have the general Formula 4 wherein a₄ and b₄ vary from 0 to 3;A₂ is a single or double bond; B₂ is selected from the group consistingof —O—, —S—, or —NR³⁸; C₂ is —CH₂ or —C═O; D₂ is selected from the groupconsisting of —O—, —S— and —NR³⁸; R³⁸ is selected from the groupconsisting of -hydrogen, C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl,C1-C10 polyalkoxyalkyl, —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20polyhydroxyalkyl, C1-C10 polyhydroxyaryl, —(CH₂)_(d)—CO₂H,—CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H, —(CH₂)_(f)—NH₂, and—CH₂—(CH₂—O—CH₂)₂—CH₂—NH₂; R⁴⁵ is a hydrogen, a halogen atom, asaccharide or a hydrophilic peptide; R⁴⁶ to R⁵⁷ are hydrogens; W⁴ and X⁴may be the same or different and are selected from the group consistingof —C(CH₃)₂, C((CH₂)_(zz)OH)CH₃, C((CH₂)_(zz)OH)₂, ((CH₂)_(zz)CO₂H)CH₃,C((CH₂)_(zz)CO₂H)₂, C((CH₂)_(zz)NH₂)CH₃, C((CH₂)_(zz)NH₂)₂,C((CH₂)_(zz)NR_(pp)R_(pz))CH₃ and C((CH₂)_(zz)NR_(pp)R_(pz))_(2;) Y⁴ andZ⁴ may be the same or different and are selected from the groupconsisting of —(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)_(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R_(pp) and R_(pz) arethe same or different and are selected from —(CH₂)_(c)—CO₂H,—CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H, —(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R¹⁴ and R¹⁵ may be thesame or different and are selected from the group consisting of-hydrogen, C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl, C1-C10polyalkoxyalkyl, —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20 polyhydroxyalkyl,C1-C10 polyhydroxyaryl, —(CH₂)_(d)—CO₂H, —CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H,—(CH₂)_(f)—NH₂, and —CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, i, and zzvary from 1 to 5; and d, f and j vary from 1 to 100.

In another preferred embodiment, the dyes according to the presentinvention have the general Formula 5 wherein a₅ varies from 0 to 3; A₃is a single or double bond; B₃ is selected from the group consisting of—O—, —S— and —NR³⁸; C₃ is —CH₂ or —C═O; D₃ is selected from the groupconsisting of —O—, —S— and —NR³⁸; R³⁸ is selected from the groupconsisting of -hydrogen, C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl,C1-C10 polyalkoxyalkyl, —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20polyhydroxyalkyl, C1-C10 polyhydroxyaryl, —(CH₂)_(d)—CO₂H,—CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H, —(CH₂)_(f)NH₂, and—CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; R⁵⁸ is a hydrogen, a halogen atom, asaccharide or a hydrophilic peptide; R⁵⁹ to R⁶⁶ are hydrogens; W⁵ and X⁵may be the same or different and are selected from the group consistingof —C(CH₃)₂, C((CH₂)_(zz)OH)CH₃, C((CH₂)_(zz)OH)₂, C((CH₂)_(zz)CO₂H)CH₃,C((CH₂)_(zz)CO₂H)₂, C((CH₂)_(zz)NH₂)CH₃, C((CH₂)_(zz)NH₂)₂,C((CH₂)_(zz)NR_(pp)R_(pz))CH₃, and C((CH₂)_(zz)NR_(pp)R_(pz))_(2;) Y⁵and Z⁵ may be the same or different and are selected from the groupconsisting of —(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O——CH₂)_(j)—CH₂—CO₂H; R_(pp) and R_(pz) arethe same or different and are selected from —(CH₂)_(c)—CO₂H,—CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H, —(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R¹⁴ and R¹⁵ may be thesame or different and are selected from the group consisting of-hydrogen, C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl, C1-C10polyalkoxyalkyl, —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20 polyhydroxyalkyl,C1-C10 polyhydroxyaryl, —(CH₂)_(d)—CO₂H, —CH₂(CH₂—O—CH₂)_(e)—CH₂—CO₂H,—(CH₂)_(f)NH₂, and —CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, i, and zzvary from 1 to 5; and d, f and j vary from 1 to 100.

In another preferred embodiment, the dyes according to the presentinvention have the general Formula 6 wherein a₆ varies from 0 to 3; A₄is a single or double bond; B₄ is selected from the group consisting of—O—, —S— and —NR³⁸; C₄ is —CH₂ or —C═O; D₄ is selected from the groupconsisting of —O—, —S— and —NR³⁸; R³⁸ is selected from the groupconsisting of -hydrogen, C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl,C1-C10 polyalkoxyalkyl, —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20polyhydroxyalkyl, C1-C10 polyhydroxyaryl, —(CH₂)_(d)—CO₂H,—CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H, —(CH₂)_(f)—NH₂, and—CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; R⁶⁷ is a hydrogen, a halogen atom, asaccharide or a hydrophilic peptide; R⁶⁸ to R⁷⁹ are hydrogens; W⁶ and X⁶may be the same of different and are selected from the group consistingof —C(CH₃)₂, C((CH₂)_(zz)OH)CH₃, C((CH₂)_(zz)OH)₂,C((CH₂)_(zz)CO₂H)CH_(3 ,) C((CH₂)_(zz)CO₂H)₂, C((CH₂)_(zz)NH₂)CH₃,C((CH₂)_(zz)NH₂)₂, C((CH₂)_(zz)NR_(pp)R_(pz))CH₃ andC((CH₂)_(zz)NR_(pp)R_(pz))₂; Y⁶ and Z⁶ may be the same or different andare selected from the group consisting of —(CH₂)_(c)—CO₂H,—CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H, —(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R_(pp) and R_(pz) arethe same or different and are selected from the group consisting of—(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R¹⁴ and R¹⁵ may be thesame or different and are selected from the group consisting of-hydrogen, C1-C10 alkyl, C1-C10 aryl, C1-C10 alkoxyl, C1-C10polyalkoxyalkyl, —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, C1-C20 polyhydroxyalkyl,C1-C10 polyhydroxyaryl, —(CH₂)_(d)—CO₂H, —CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H,—(CH₂)_(f)—NH₂, and —CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, i, and zzvary from 1 to 5; and d, f and j vary from 1 to 100.

The compositions of the invention can be formulated into diagnosticcompositions for enteral or parenteral administration. Thesecompositions contain an effective amount of the dye along withconventional pharmaceutical carriers and excipients appropriate for thetype of administration contemplated. For example, parenteralformulations advantageously contain a sterile aqueous solution orsuspension of dye according to this invention. Parenteral compositionsmay be injected directly or mixed with a large volume parenteralcomposition for systemic administration. Such solutions also may containpharmaceutically acceptable buffers and, optionally, electrolytes suchas sodium chloride.

Formulations for enteral administration may vary widely, as is wellknown in the art. In general, such formulations are liquids whichinclude an effective amount of the dye in aqueous solution orsuspension. Such enteral compositions may optionally include buffers,surfactants, thixotropic agents, and the like. Compositions for oraladministration may also contain flavoring agents and other ingredientsfor enhancing their organoleptic qualities.

The diagnostic compositions are administered in doses effective toachieve the desired enhancement. Such doses may vary widely, dependingupon the particular dye employed, the organs or tissues which are thesubject of the imaging procedure, the imaging equipment being used, andthe like.

The diagnostic compositions of the invention are used in theconventional manner. The compositions may be administered to a patient,typically a warm-blooded animal, either systemically or locally to theorgan or tissue to be imaged, and the patient then subjected to theimaging procedure.

A combination of the above represents an important approach to thesynthesis and use of novel cyanine and indocyanine dyes with a varietyof photophysical and chemical properties. The dyes of this invention arenew and they are useful for biomedical applications. The presentinvention is further detailed in the following Examples, which areoffered by way of illustration and are not intended to limit the scopeof the invention in any manner. Reliable computational methods for theprediction of the absorption maxima of some of the dyes were alsoestablished. Standard techniques well known in the art or the techniquesspecifically described below are utilized.

EXAMPLE 1

Synthesis of Bis(ethylcarboxymethyl)indocyanine Dye (Scheme 1, R₁,R₂=fused phenyl; A=CH₂, n=1 and R=R′=CO₂H)

A mixture of 1,1,2-trimethyl-[1H]-benz[e]indole (9.1 g, 43.58 mmoles)and 3-bromopropanoic acid (10.0 g, 65.37 mmoles) in 1,2-dichlorobenzene(40 mL) was heated at 110° C. for 12 hours. The solution was cooled toroom temperature and the red residue obtained was filtered and washedwith acetonitrile:diethyl ether (1:1) mixture. The solid obtained wasdried under vacuum to give 10 g (64%) of light brown powder. A portionof this solid (6.0 g; 16.56 mmoles), glutaconaldehyde dianilmonohydrochloride (2.36 g, 8.28 mmoles) and sodium acetate trihydrate(2.93 g, 21.53 mmoles) in ethanol (150 mL) were refluxed for 90 minutes.After evaporating the solvent, 40 mL of a 2 N aqueous HCI was added tothe residue and the mixture was centrifuged and the supernatant wasdecanted. This procedure was repeated until the supernatant becamenearly colorless. About 5 mL of water:acetonitrile (3:2) mixture wasadded to the solid residue and lyophilized to obtain 2 g of dark greenflakes. The purity of the compound was established with ¹H-NMR andLC-Mass spectrometry.

EXAMPLE 2

Synthesis of Bis(pentylcarboxymethyl)indocyanine Dye

(FIG. 1, R₁=fused phenyl; A=CH₂, n=4 and R=R′=CO₂H)

A mixture of 1,1,2-trimethyl-[1 H]-benz[e]indole (20 g, 95.6 mmoles) and6-bromohexanoic acid (28.1 g, 144.1 mmoles) in 1,2-dichlorobenzene (250mL) was heated at 110 C for 12 hours. The green solution was cooled toroom temperature and the brown solid precipitate formed was collected byfiltration. After washing the solid with 1,2-dichlorobenzene and diethylether, the brown powder obtained (24 g, 64%) was dried under vacuum atroom temperature. A portion of this solid (4.0 g; 9.8 mmoles),glutaconaldehyde dianil monohydrochloride (1.4 g, 5 mmoles) and sodiumacetate trihydrate (1.8 g, 12.9 mmoles) in ethanol (80 mL) were refluxedfor 1 hour. After evaporating the solvent, 20 mL of a 2 N aqueous HCIwas added to the residue and the mixture was centrifuged and thesupernatant was decanted. This procedure was repeated until thesupernatant became nearly colorless. About 5 mL of water:acetonitrile(3:2) mixture was added to the solid residue and lyophilized to obtainabout 2 g of dark green flakes. The purity of the compound wasestablished with ¹ H-NMR and LC-Mass spectrometry.

EXAMPLE 3

Synthesis of Bisethylcarboxymethylindocyanine Dye

(FIG. 1, R₁=R₂=H; A=CH₂, n=1 and R=R′=CO₂H)

This compound was prepared as described in Example 1 except that1,1,2-trimethylindole was used as the starting material.

EXAMPLE 4

Synthesis of Bis(hexaethyleneglycolcarboxymethyl)indocyanine Dye

(FIG. 1, R₁=R₂=fused phenyl; A=CH₂OCH₂, n=6 and R=R′=CO₂H)

This compound was prepared as described in Example 1 except that(ω-bromohexaoxyethyleneglycolpropiolic acid was used in place ofbromopropanoic acid and the reaction was carried out in1,2-dimethoxypropane.

EXAMPLE 5

Synthesis of Bisethylcarboxymethylindocyanine Dye

(FIG. 2, R₁=R₂=fused phenyl; A=CH₂, and n=0)

A solution of 50 ml of dimethylformamide and benzyl bromoacetate (16.0g, 70 mmol) was stirred in a 100 ml three-neck flask. Solid potassiumbicarbonate (7.8 g, 78 mmol) was added. The flask was purged with argonand cooled to 0° C. with an ice bath. To the stirring mixture was addeddropwise a solution of ethanolamine (1.9 g, 31 mmol) and 4 ml ofdimethylformamide over 5 minutes. After the addition was complete themixture was stirred for 1 hour at 0° C. The ice bath was removed and themixture stirred at room temperature over night. The reaction mixture waspartitioned between 100 ml of methylene chloride and 100 ml of saturatedsodium bicarbonate solution. The layers were separated and the methylenechloride layer was again washed with 100 ml of saturated sodiumbicarbonate solution. The combined aqueous layers were extracted twicewith 25 ml of methylene chloride. The combined methylene chloride layerswere washed with 100 ml of brine, and dried over magnesium sulfate. Themethylene chloride was removed with aspirator vacuum at ca. 35° C., andthe remaining dimethylformamide was removed with vacuum at about 45° C.The crude material was left on a vacuum line over night at roomtemperature.

The crude material from above was dissolved in 100 ml of methylenechloride at room temperature. Triphenylphosphine (8.91 g, 34 mmol) wasadded and dissolved with stirring. An argon purge was started and themixture cooled to 0° C. with an ice bath. The N-bromosuccinimide (6.05g, 34 mmol) was added portionwise over 2 minutes. The mixture wasstirred for 1.5 hours at 0° C. The methylene chloride was removed withvacuum and gave a purple oil. This oil was triturated with 200 ml ofether with constant manual stirring. During this time the oil becamevery thick. The ether solution was decanted and the oil was trituratedwith 100 ml of ether. The ether solution was decanted and the oil wasagain triturated with a 100 ml portion of ether. The ether was decantedand the combined ether solutions allowed to stand for about 2 hours toallow the triphenylphosphine oxide to crystallize. The ether solutionwas decanted from the crystals and the solid washed with 100 ml ofether. The volume of the combined ether abstracts was reduced withvacuum until a volume of about 25 ml was obtained. This was allowed tostand over night at 0° C. Ether (10 ml) was added to the cold mixturewhich was mixed to suspend the solid. The mixture was percolated througha column of 45 g of silica gel and eluted with ether, 75 ml fractionswere collected. The fractions that contained product by TLC were pooledand the ether removed with vacuum. This gave 10.1 g of crude product.The material was flash chromatographed on silica gel with hexane,changing to 9:1 hexane:ether. The product-containing fractions werepooled and the solvents removed with vacuum. This gave 7.4 g (57% yield)of pure product.

A mixture of 10% palladium on carbon (1 g) and a solution of the benzylester (10 g) in 150 ml of methanol was hydrogenolyzed at 25 psi for 2hours. The mixture was filtered over celite and the residue was washedwith methanol. The solvent was evaporated to give a viscous oil inquantitative yield.

Reaction of the bromide with 1,1,2-trimethyl-[1 H]-benz[e]indole wascarried out as described in Example 1.

EXAMPLE 6

Bis(ethylcarboxymethyldihydroxyl)indocyanine Dye (FIG. 3)

The hydroxy-indole compound is readily prepared by literature method (P.L. Southwick, J. G. Cairns, L. A. Ernst, and A. S. Waggoner, One potFischer synthesis of (2,3,3-trimethyl-3-H-indol-5-yl)acetic acidderivatives as intermediates for fluorescent biolabels. Org. Prep.Proced. Int. Briefs, 1988, 20(3), 279-284). Reaction ofp-carboxymethylphenylhydrazine hydrochloride (30 mmol, 1 equiv.) and1,1-bis(hydroxymethyl)propanone (45 mmole, 1.5 equiv.) in acetic acid(50 mL) at room temperature for 30 minutes and at reflux for 1 gives(3,3-dihydroxymethyl2-methyl-3-H-indol-5-yl)-acetic acid as a solidresidue. The reaction of 3-bromopropyl-N,N-bis(carboxymethyl)amine,which was prepared as described in Example 5, with the intermediateindole and subsequent reaction of the indole intermediate withglutaconaldehyde dianil monohydrochloride (see Example 1) gives thedesired product.

EXAMPLE 7

Synthesis of Bis(propylcarboxymethyl)indocyanine Dye (FIG. 4)

The intermediate 2-chloro-1-formyl-3-hydroxymethylenecyclohexane wasprepared as described in the literature (G. A. Reynolds and K. H.Drexhage, Stable heptamethine pyrylium dyes that absorb in the infrared.J. Org. Chem., 1977, 42(5), 885-888). Equal volumes (40 mL each) ofdimethylformamide (DMF) and dichloromethane were mixed and the solutionwas cooled to −10° C. in acetone-dry ice bath. Under argon atmosphere,phosphorus oxychloride (40 mL) in dichloromethane was added dropwise tothe cool DMF solution. The resulting solution was allowed to warm up toroom temperature and refluxed for 6 hours. After cooling to roomtemperature, the mixture was poured into ice-cold water and stored at 4°C. for 12 hours. About 8 g of yellow powder was obtained afterfiltration. Condensation of the cyclic dialdehyde with the indoleintermediate is carried out as described in Example 1. Further thefunctionalization of the dye with bis (isopropylidene acetal protectedmonosaccharide by the method described in the literature (J. H.Flanagan, C. V. Owens, S. E. Romero, et al., Near infraredheavy-atom-modified fluorescent dyes for base-calling in DNA-sequencingapplication using temporal discrimination. Anal. Chem., 1998, 70(13),2676-2684).

EXAMPLE 8

Synthesis of Bis(ethylcarboxymethyl)indocyanine Dye (FIG. 5) These dyesare prepared as described in Experiment 7. These dyes absorb in theinfrared region. The typical example shown in FIG. 5 have estimatedabsorption maximum at 1036 nm.

EXAMPLE 9

Synthesis of Peptides

The procedure described below is for the synthesis of Octreotate. Otherpeptides were prepared by a similar procedure with minor modificationsin some cases. These peptides were used to illustrate the ease of usingdyes of this invention to prepare bioconjugates.

The octapeptide was prepared by an automated fluorenylmethoxycarbonyl(Fmoc) solid phase peptide synthesis using a commercial peptidesynthesizer from Applied Biosystems (Model 432A SYNERGY PeptideSynthesizer). The first peptide cartridge contained Wang resinpre-loaded with Fmoc-Thr on a 25 μmole scale. Subsequent cartridgescontained Fmoc-protected amino acids with side chain protecting groupsfor the following amino acids: Cys(Acm), Thr(t-Bu), Lys(Boc), Trp(Boc)and Tyr(t-Bu). The amino acid cartridges were placed on the peptidesynthesizer and the product was synthesized from the C- to theN-terminal position. The coupling reaction was carried out with 75μmoles of the protected amino acids in the presence of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-hydroxybenzotriazole (HOBt). The Fmoc protecting group wasremoved with 20% piperidine in dimethylformamide. After the synthesiswas complete, the thiol group was cyclized with thalliumtrifluoroacetate and the product was cleaved from the solid support witha cleavage mixture containing trifluoroacetic acid (85%):water(5%):phenol (5%):thioanisole (5%) for 6 hours. The peptide wasprecipitated with t-butyl methyl ether and lyophilized withwater:acetonitrile (2:3) mixture. The peptide was purified by HPLC andanalyzed with LC/MS. The amino acid sequence of Octreotate is:D-Phe-Cys′Tyr-D-Trp-Lys-Thr-Cys′-Thr, wherein Cys′ indicates thepresence of an intramolecular disulfide bond between two cysteine aminoacids.

Octreotide was prepared by the same procedure:D-Phe-Cys′-Tyr-D-Trp-Lys-Thr-Cys′-Thr-OH, wherein Cys′ indicates thepresence of an intramolecular disulfide bond between two cysteine aminoacids.

Bombesin analogs were prepared by the same procedure except thatcyclization with thalium trifluoroacetate was not needed. Side-chaindeprotection and cleavage from the resin was carried out with 50 μL eachof ethanedithiol, thioanisole and water, and 850 μL of trifluoroaceticacid . Two analogues were prepared:Gly-Ser-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂andGly-Asp-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂.

Cholecystokinin octapeptide analogs were prepared as described forOctreotate without the cyclization step. Three analogs were prepared:Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH₂;Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH₂;D-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH₂.

Neurotensin analog was prepared as described for Octreotate without thecyclization step: D-Lys-Pro-Arg-Arg-Pro-Tyr-lle-Leu.

EXAMPLE 10

Synthesis of Peptide-Dye Conjugates

The method described below is for the synthesis of Octreotate conjugatesbut a similar procedure is used for the synthesis of other peptide-dyeconjugates.

The Octreotate was prepared as described in Example 6 but the peptidewas not cleaved from the solid support and the N-terminal Fmoc group ofPhe was retained. The thiol group was cyclized with thalliumtrifluoroacetate and the Phe was deprotected to liberate the free amine.Bisethylcarboxymethylindocyanine dye (53 mg, 75 μmoles) A was added toan activation reagent consisting of a 0.2 M solution of HBTU/HOBt inDMSO (375 μL), and 0.2 M solution of diisopropylethylamine in DMSO (375μL). The activation was complete in about 30 minutes and the resin-boundpeptide (25 μmoles) was added to the dye. The coupling reaction wascarried out at room temperature for 3 hours. The mixture was filteredand the solid residue was washed with DMF, acetonitrile and THF. Afterdrying the green residue, the peptide was cleaved from the resin and theside chain protecting groups were removed with a mixture of 85%trifluoroacetic acid, 2.5% water, 2.5% thioanisole and 2.5% phenol. Theresin was filtered and cold t-butyl methyl ether (MTBE) was used toprecipitate the dye-peptide conjugate which was dissolved inacetonitrile:water (2:3) mixture and lyophilized. The product waspurified by HPLC to give themonoOctreotate-Bisethylcarboxymethylindocyanine dye (Cytate 1, 80%) andthe bisOctreotate-Bisethylcarboxymethylindocyanine dye (Cytate 2, 20%).The monoOctreotate conjugate can be obtained almost exclusively (>95%)over the bis conjugate by reducing the reaction time to 2 hours.However, this also leads to incomplete reaction and the free Octreotatemust be carefully separated from the dye conjugate in order to avoidsaturation of the receptors by the non-dye conjugated peptide.

Octreotate-bispentylcarboxymethylindocyanine dye was prepared asdescribed above with some modifications.Bispentylcarboxymethylindocyanine dye (60 mg, 75 μmoles) was added to anactivation reagent consisting of a 0.2 M solution of HBTU/HOBt in DMSO(400 μL), and 0.2 M solution of diisopropylethylamine in DMSO (400 μL).The activation was complete in about 30 minutes and the resin-boundpeptide (25 μmoles) was added to the dye. The reaction was carried outat room temperature for 3 hours. The mixture was filtered and the solidresidue was washed with DMF, acetonitrile and THF. After drying thegreen residue, the peptide was cleaved from the resin and the side chainprotecting groups were removed with a mixture of 85% trifluoroaceticacid, 2.5% water, 2.5% thioanisole and 2.5% phenol. The resin was Mfiltered and cold t-butyl methyl ether (MTBE) was used to precipitatethe dye-peptide conjugate which was dissolved in acetonitrile:water(2:3) mixture and lyophilized. The product was purified by HPLC to giveOctreotate-1,1,2-trimethyl-[1H]-benz[e]indole propanoic acid conjugate(10%), monoOctreotate-bispentylcarboxymethylindocyanine dye (Cytate 3,60%) and bisOctreotate-bispentylcarboxymethylindocyanine dye (Cytate 4,30%).

While the invention has been disclosed by reference to the details ofpreferred embodiments of the invention, it is to be understood that thedisclosure is intended in an illustrative rather than in a limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, within the spirit of the invention and thescope of the appended claims.

8 1 8 PRT Artificial Sequence Description of ArtificialSequenceOctreotide 1 Phe Cys Tyr Trp Lys Thr Cys Thr 1 5 2 11 PRTArtificial Sequence SITE (11) This C-terminal amino acid ends with anNH2 2 Gly Ser Gly Gln Trp Ala Val Gly His Leu Met 1 5 10 3 11 PRTArtificial Sequence SITE (11) THIS C-TERMINAL AMINO ACID ENDS WITH ANNH2. 3 Gly Asp Gly Gln Trp Ala Val Gly His Leu Met 1 5 10 4 8 PRTArtificial Sequence SITE (8) THIS C-TERMINAL RESIDUE ENDS WITH NH2. 4Asp Tyr Met Gly Trp Met Asp Phe 1 5 5 8 PRT Artificial Sequence MOD_RES(3) Nle 5 Asp Tyr Xaa Gly Trp Xaa Asp Phe 1 5 6 8 PRT ArtificialSequence MOD_RES (3) Nle 6 Asp Tyr Xaa Gly Trp Xaa Asp Phe 1 5 7 8 PRTArtificial Sequence SITE (1) THIS IS D-LYSINE. 7 Lys Pro Arg Arg Pro TyrIle Leu 1 5 8 8 PRT Artificial Sequence Description of ArtificialSequenceOctreotate 8 Phe Cys Tyr Trp Lys Thr Cys Thr 1 5

What is claimed is:
 1. A method for restoring in vivo or in vitrofluorescence by adding one to fifty percent biocompatible organicsolvents to the diagnostic or therapeutic compositions of dye moleculeswherein said dye molecules are represented by the formula:

wherein a₄ and b₄ are independently from 0 to 5; W⁴ and X⁴ areindependently selected from the group consisting of (C((CH₂)_(zz)OH)₂,C((CH₂)_(zz)CO₂H)₂, C((CH₂)_(zz)NR_(pp)R_(pz))₂; and zz is from 1 to 5;R_(pp) and R_(pz) are selected from the group consisting of—(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; Y⁴ and Z⁴ areindependently selected from the group consisting of —(CH₂)_(c)—CO₂H,—CH₂—(CH₂—O—CH₂)_(d)—CH₂CO₂H, —(CH₂)_(e)—NH₂,—CH₂—(CH₂—O—CH₂)_(f)—CH₂—NH₂, —(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; A₂ is a single or adouble bond; if A₂ is a single bond, then B₂ is independently selectedfrom the group consisting of —O—, —S—, —Se—, —P— and —NR³⁸ and D₂ isCR⁴²; if A₂ is a double bond, then B₂ is selected from the groupconsisting of —O—, —S—, —Se—, —P— and —NR³⁸, C₂ is selected fromnitrogen and —CR⁴¹ and D₂ is —CR⁴²; R⁴⁵ to R⁵⁷ are independentlyselected from the group consisting of hydrogen, C1-C10 alkyl C1-C10alkoxyl, cyano, nitro, halogen, and —NR⁴³R⁴⁴; R R^(14,) R¹⁵ and R³⁸ toR⁴² are selected from the group consisting of -hydrogen, C1-C10 alkyl,C1-C10 alkoxyl —CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, —(CH₂)_(d)—CO₂H,—CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H, —(CH₂)_(f)—NH₂, and—CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂; c, e, g, h, and i are independently from 1to 10; d, f and j are independently from 1 to 100; and R⁴³ and R⁴⁴ areselected from the group consisting of hydrogen and C1-C10 alkyl.
 2. Themethod of claim 1 wherein said dye molecule is dissolved in a mediumcomprising one to 50 percent dimethyl sulfoxide (DMSO).
 3. Thecomposition of claim 1 wherein a₄ and b₄ are independently from 0 to 3;A₂ is a single bond; B₂ is selected from the group consisting of —O—,—S—, and —NR³⁸; C₂ is selected from —CH₂; D₂ is CR⁴²; R⁴⁶ to R⁵⁷ arehydrogens; W⁴ and X⁴ are independently selected from the groupconsisting of C((CH₂)_(zz)OH)₂, C((CH₂) _(zz)CO₂H)₂, andC((CH₂)_(zz)NR_(pp)R_(pz))₂; Y⁴ and Z⁴ are independently selected fromthe group consisting of —(CH₂)_(c)—CO₂H, —CH₂—(CH₂—O—CH₂)_(d)—CH₂—CO₂H,—(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂)_(j)—CH₂—CO₂H; R_(pp) and R_(pz) areindependently selected from —(CH₂)_(c)—CO₂H,—CH₂—(CH₂—O—CH₂)_(d)CH₂—CO₂H, —(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H, and—(CH₂)_(i)—N(R¹⁵)—CH₂—(CH₂—O—CH₂ _(j)—CH₂—CO₂H; c, e, g, h, i, and zzare independently from 1 to 5; d, f and j are independently from 1 to100; and R¹⁴ and R¹⁵ are independently selected from the groupconsisting of hydrogen, C1-C10 alkyl, C1-C alkoxyl,—CH₂(CH₂—O—CH₂)_(c)—CH₂—OH, —(CH₂)_(d)CO₂H,—CH₂—(CH₂—O—CH₂)_(e)—CH₂—CO₂H, —(CH₂)_(f)—NH₂, and—CH₂—(CH₂—O—CH₂)_(g)—CH₂—NH₂.
 4. The composition of claim 1 wherein a₄and b₄ are 3; A₂ is a double bond; B₂ is —O—; C₂ and D₂ are CH; R⁴⁶ toR⁵⁷ are hydrogens; W⁴ and X⁴ are independently selected from the groupconsisting of -C((CH₂)_(zz)OH)₂, C((CH₂)_(zz)CO₂H)₂ andC((CH₂)_(zz)NR_(pp)R_(pz))₂; Y⁴ and Z⁴ are the same and are selectedfrom the group consisting of —(CH₂)_(c)CO₂H, —CH₂CH₂—O—CH₂)_(d)—CH₂—CO₂Hand —(CH₂)_(g)—N(R¹⁴)—(CH₂)_(h)—CO₂H.